Commerical production of proteases in plants

ABSTRACT

Production of proteases in plants is set forth, whereby heterologous DNA encoding the protease is introduced into the plant and expression of the protein achieved. By such methods, expression is achieved in plants wherein the plant cell is not damaged, the protein can be recovered without contamination by other proteases, and can be expressed at levels such that commercial production of the enzyme is obtained. Expression levels can be at 0.1% of total soluble protein of the plant, or higher.

STATEMENT OF RELATIONSHIP TO PRIOR APPLICATION

This patent application is a continuation of previously filed andco-pending U.S. patent application Ser. No. 09/569,284 filed May 12,2000, which is a continuation of previously filed and co-pending U.S.patent application Ser. No. 09/120,582, filed Jul. 22, 1998, now U.S.Pat. No. 6,087,558.

BACKGROUND OF THE INVENTION

Commercial production of proteases that is high yielding, economical andprovides ease in manufacture and processing would provide considerableadvantages to many industries. Proteases are used in a variety ofcommercial applications, including pharmaceutical uses, medicalprocesses, lab processes, in sequencing amino acids, among others. Manyproteases used in such commercial applications are obtained from sourcesthat are difficult and costly to maintain, that are not high yielding,and include undesirable contaminants.

Among the problems encountered are that either animal organs or bacteriaare the common sources for proteases. For example, pepsin is obtainedfrom gastric mucosa, carboxypeptidase A and B are obtained from thepancreas of animals, and leucine aminopeptidase from the kidney andintestinal mucosa. Contamination by undesirable components produced bythe animal cells can impact the final product. Bacterial sourcestypically cannot produce the protease in reliable or sufficient quantityto be useful for commercial purposes. An example of proteases obtainedfrom bacteria include subtilisin and themolysin, obtained from strainsof Bacillus. As noted by James Wells and David Estell, “Most enzymes areexpressed in minute amounts, and no generic solution is available forthe expression of large amounts of an active enzyme from its clonedgene.” Wells, J. and Estell, D. “Subtilisin: An Enzyme Designed to BeEngineered” Trends in Biochemical Sciences (a review) vol. 13 pp.291-297.

By way of example, in the digestive process, a hormone action istriggered that releases digestive juice made by the pancreas. This juicecontains several precursors or zymogens, including trypsinogen, andchymotrypsinogen, among others. Trypsinogen is a protein which is theprecursor or zymogen molecule of trypsin. By action of enterokinase,which removes a hexapeptide from the NH₂-terminal end of the trypsinogenmolecule, trypsin is formed. Trypsin is a protease which hydrolyzes thepeptide bonds of the oligopeptides in the intestine by cleaving on thecarboxyl side of lysine and arginine residues. Trypsin also activateschymotrypsinogen to chymotrypsin. Chymotrypsin hydrolyzes the peptidebonds involving phenylalanine, tyrosine and tryptophan.

Trypsin has a number of uses in the biological sciences and in themedical field An example is the use of trypsin in identifying thesequence of amino acids. It is useful in many processes where its'selective cleaving can be employed. For example, because its' cleavageis specific to select amino acids, it can be used to break down apolypeptide into fragments of known number. Importantly, this substratespecificity is also useful in converting biosynthetically producedmolecules to preferred molecules. It is used in this manner to convertproinsulin to insulin by removal of the connecting peptide. Thus,trypsin has many commercially valuable uses.

The current source of trypsin is the organs of animals, with bovine andporcine pancreas the primary common source of the enzyme. There arenumerous difficulties associated with obtaining trypsinogen or trypsinfrom these sources. One is that there is considerable contamination byother proteases. Chymotrypsin is one of the additional proteases in thecontaminants that may cleave the product in an undesired manner.

Further, there are obvious expenses and handling concerns when shippingand using animal pancreas. They must be fresh, kept sterile, shipped ina manner to maintain freshness of the organ, and require special storagespace of sufficient size to accommodate the animal organs. It alsorequires the care, feeding, and slaughtering of the animals that are thesource. Additionally, some users of the end product have concerns aboutuse of enzymes prepared from animal sources as components in humanproducts.

Prior attempts to avoid these problems have included expressingtrypsinogen in bacteria. A European patent application describesintroducing trypsin and trypsinogen into E coli, and selecting fortransformed bacteria by use of an antibiotic resistance marker. SeeGreaney, EP 0 587 681. They used a variety of E. coli host cells in theconstruction of vectors and expression systems. There, the inventorsreported that while some bacterial strains expressed the protein, otherswould not.

The present invention overcomes these obstacles by providing for a planthaving a heterologous DNA sequence that expresses a protease. In onepreferred embodiment, the protease is trypsin or trypsinogen. Theinventors have been able to achieve expression at commerciallyacceptable levels of production and provide for a homogeneous productwhich does not contain the contaminants associated with animal sources.Since the source is plant, other undesirable proteases produced in thehost are not a problem. The inventors have also found there areadvantages to expressing proteases in seed. The seed is rich in proteaseinhibitors, thus enhancing stability. This is an even further advantagewhen the protease is not the inactive zymogen, (such as trypsinogen) butthe active protease (such as trypsin). The storage, shipping and expenseassociated with expressing the enzyme in plants is vastly superior tothe animal or bacterial sources.

Thus, it is an object of the invention to provide for plants and plantcells having DNA comprising heterologous nucleotide sequences encoding aprotease.

It is a further object of this invention to provide for expression ofproteases at commercially acceptable levels.

Another object of the invention is to provide for production ofproteases which is not contaminated with animal proteases and othercontaminants.

Yet another object of the invention is to provide for production ofproteases in seed.

A still further object of the invention is to provide for a method ofproduction of proteases in a biomass of plants.

Another object of the invention is to provide for production ofproteases which is economical and provides for ease in production andhandling.

A further object of the invention is to provide for production oftrypsin or trypsinogen in plants at commercially acceptable levels andwithout contamination from animal proteases.

These and the other objectives will become apparent by the descriptionbelow.

All references cited herein are incorporated herein by reference.

SUMMARY OF THE INVENTION

The invention is production of proteases in plants at commerciallyacceptable levels by introducing into a plant, DNA encoding aheterologous nucleotide sequence encoding the protease. In a preferredembodiment of the invention, the DNA is linked to a promoter such thatexpression levels of 0.1% and higher may be achieved. Yet anotherembodiment provides for the protease to be trypsin or trypsinogen.

In another preferred embodiment of the invention, the protease isexpressed in the seed of the plant. In one embodiment, the promoter maybe ubiquitin. In another preferred embodiment, the protease encoding DNAis linked with a seed specific promoter, such as the phaseolin promoter.Another preferred aspect of the invention provides for use of a signalsequence in the DNA construct, and in yet another preferred embodiment,may be the barley alpha amylase signal sequence.

In accordance with a second aspect of the invention, a method ofreproducing proteases in commercial quantities is provided by providinga biomass from a plurality of plants, of which at least certain plantscontain a DNA molecule comprised of a heterologous nucleotide sequenceencoding the protease, wherein the nucleotide sequence is operablylinked to a promoter to effect expression of the protease by thoseplants. The plant or plant tissue can then be fed directly to theanimal, or the protein extracted. Where the protein is used as adigestive enzyme it may be particularly advantageous to feed the plantto the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows p8244. In p8244, the trypsinogen gene is driven by thephaseolin promoter, and contains the brazil nut protein signal sequence,with the trypsinogen gene followed by the phaseolin terminator. A repeatof these sequences follows, with the selectable marker PAT gene drivenby the CaMV35S promoter, and followed by the 35S terminator. The rightand left borders of the T-DNA and replication origin sequences necessaryfor incorporation into the plant using Agrobacteriu, are also included.

FIG. 2 shows p4347. In p4347, the trypsinogen gene is driven by theubiquitin promoter which includes the first exon and intron, the barleyalpha amylase signal sequence, and the trypsinogen gene followed by aPinII transcription termination sequence. The selectable marker, themoPAT gene is controlled by the CaMV35S promoter and terminator regions.The plasmid includes sequences necessary for incorporation into theplant using Agrobacterium, including the left and right borders of theT-DNA, the replication origin region, the co-integration site andspectinomycin resistance sequences as the selection agent.

FIG. 3 shows p5443. It is essentially the same as p4347, except thattandem repeats of the trypsinogen gene are included.

FIGS. 4A and 4B are Western gel blots of protein extracted fromtransformed canola seed expressing trypsinogen, and controls.

FIGS. 5A and 5B are Southern gel blots of DNA extracted from transformedcanola plants expressing trypsinogen in the seed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have determined that commercial production ofproteases in plants is not only feasible but also offers substantialadvantages over the conventional approach of obtaining the protein fromanimal organs. Plant tissue as a raw material would be cheaper and morestable to store. Contamination by proteases that have an undesiredimpact on the product can be avoided, since they are not produced froman animal source. Use from a plant source also avoids concerns aboutproducts from animal sources applied in products for human use.

In accordance with the present invention, a DNA molecule comprising atransformation/expression vector is engineered to incorporateprotease-encoding DNA. By protease-encoding DNA, it is also meant toinclude both the protease in its active form, and the inactive formwhich can then be activated. In an embodiment of the invention, the DNAmolecule encodes trypsin (the protease) or trypsinogen (the inactiveform). Trypsin and trypsinogen differ in that trypsin is missing thefirst six amino acids of trypsinogen. If trypsin were placed in theplant, like any protease, there is a high likelihood that it woulddigest plant proteins, resulting in cell death. This can be prohibitedby expressing trypsin in the seed of the plant, which is rich inprotease inhibitors. Trypsinogen is the pro-form of the enzyme, and notan active protease, thus allowing for expression of the zymogen withoutdestroying its host. The proenzyme can later be readily converted totrypsin by cleaving the first six amino acids.

Genes encoding the proteins are well known. When referring to “trypsin”or “trypsinogen” it is meant to encompass fragments and variations ofthe protein that still retain the characteristics of the proteolyticenzyme, and all genes which encode the proteins that fall within thecategory or trypsin or trypsinogen. In one example, Greaney, supra,discloses chemically synthesized genes for both trypsin and trypsinogen.The gene used in the examples below was made publicly available throughGenbank, as accession number P00760. Isolation and cloning of thetrypsinogen molecule can be accomplished by one skilled in the art usingstandard methodology.

Therefore, a gene for use in the present invention can be subcloned in avector of choice. In another example, it is possible to screen a cDNAlibrary with anti-protease antibodies. The known methodologies usedwould include identification of the gene by hybridization with probes,PCR, probe/promoter/synthetic gene synthesis, sequencing, molecularcloning and other techniques which are well known to those skilled inmolecular biology. While it is possible to synthesize the gene toreflect preferred codon usage in plants, and may be useful in increasingexpression of select proteases, (See, Murray et al, Nucleic Acid Res.17:477-498 (1980)), it may not be necessary in all cases, as was foundwith the gene used in the examples below.

A problem associated with expressing proteases, such as trypsin, in aplant is that since it is a protease it may digest the proteins in theplant resulting in cell death. As noted, this effect is avoided bydirecting expression of the protease to seed. The protease inhibitors inthe seed inhibit this effect. In a preferred embodiment of the inventionavoiding protein degradation in the plant is further aided by secretionof the protein to the cell wall of the plant.

This may be accomplished by use of a signal sequence and in a preferredembodiment is the barley alpha amylase signal sequence. Rogers, J. Biol.Chem. 260:3731-3738 (1985), or brazil nut protein when used in canola.Use of the signal sequence can also assist in increasing expressionlevels.

Another alternative is to express the zymogen, such as trypsinogen, inthe plant, which is the pro-form of the enzyme and not a protease. Theplant's own proteases are sequestered in the vacuoles and thus will notbreak down the zymogen. Whether the protease or the zymogen isexpressed, the signal sequence provides an additional benefit inincreasing expression level of the protein. See Jones and Robinson,Tansley Review, 17:567-597 (1989). The construct is made such that asignal peptide is fused to the N-terminus of the mature proteinsequence, allowing for normal cellular processing to cleave accuratelythe protein molecule and yield mature protein.

The methods available for putting together such a relatively shortsynthetic gene comprising the various modifications for improvedexpression described above can differ in detail. However, the methodsgenerally include the designing and synthesis of overlapping,complementary synthetic oligonucleotides which are annealed and ligatedtogether to yield a gene with convenient restriction sites for cloning.The methods involved are standard methods for a molecular biologist.

Once the gene has been isolated and engineered to contain some or all ofthe features described above, it is placed into an expression vector bystandard methods. The selection of an appropriate expression vector willdepend upon the method of introducing the expression vector into hostcells. A typical expression vector contains prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancegene to provide for the growth and selection of the expression vector inthe bacterial host; a cloning site for insertion of an exogenous DNAsequence, which in this context would code for the protease; eukaryoticDNA elements that control initiation of transcription of the exogenousgene, such as a promoter; and DNA elements that control the processingof transcripts, such as transcription termination/polyadenylationsequences. It also can contain such sequences as are needed for theeventual integration of the vector into the plant chromosome.

In a preferred embodiment, the expression vector also contains a geneencoding a selection marker which is functionally linked to a promoterthat controls transcription initiation. For a general description ofplant expression vectors and reporter genes, see Gruber et al, “Vectorsfor Plant Transformation” in Methods of Plant Molecular Biology andBiotechnology 89-119 (CRC Press, 1993).

Promoter elements employed to control expression of the protease and theselection gene, respectively, can be any plant-compatible promoter.Those can be plant gene promoters, such as, for example, the ubiquitinpromoter, the promoter for the small subunit of ribulose-1,5-bis-phosphate carboxylase, or promoters from the tumor-inducingplasmids from Agrobacterium tumefaciens, such as the nopaline synthaseand octopine synthase promoters, or viral promoters such as thecauliflower mosaic virus (CaMV) 19S and 35S promoters or the figwortmosaic virus 35S promoter. See Kay et al, Science 236:1299 (1987) andEuropean patent application No. 0 342 926. See international applicationWO 91/19806 for a review of illustrative plant promoters suitablyemployed in the present invention. The range of available plantcompatible promoters includes tissue specific and inducible promoters.

In one embodiment of the present invention, the exogenous DNA is underthe transcriptional control of a plant ubiquitin promoter. Plantubiquitin promoters are well known in the art, as evidenced by Europeanpatent application no. 0 342 926.

In a further preferred embodiment, a tissue specific promoter isprovided to direct transcription of the DNA preferentially to the seed.One such promoter is the phaseolin promoter. See, Bustos et al.“Regulation of β-glucuronidase Expression in Transgenic Tobacco Plantsby an A/T-Rich cis-Acting Sequence Found Upstream of a French Beanβ-Phaseolin Gene” The Plant Cell Vol. 1, 839-853 (1989).

In another preferred embodiment, the selective gene is aglufosinate-resistance encoding DNA and in a preferred embodiment can bethe phosphinothricin acetyl transferase (“PAT”) or maize optimized PATgene under the control of the CaMV 35S promoter. The gene confersresistance to bialaphos. See, Gordon-Kamm et al, The Plant Cell 2:603(1990); Uchimiya et al, Bio/Technology 11:835 (1993), and Anzai et al,Mol. Gen. 219:492 (1989).

Obviously, many variations on the promoters, selectable markers andother components of the construct are available to one skilled in theart.

In accordance with the present invention, a transgenic plant is producedthat contains a DNA molecule, comprised of elements as described above,integrated into its genome so that the plant expresses a heterologousprotease-encoding DNA sequence. In order to create such a transgenicplant, the expression vectors containing the gene can be introduced intoprotoplasts, into intact tissues, such as immature embryos andmeristems, into callus cultures, or into isolated cells. Preferably,expression vectors are introduced into intact tissues. General methodsof culturing plant tissues are provided, for example, by Miki et al,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick et al (eds) pp. 67-68 (CRCPress 1993) and by Phillips et al, “Cell/Tissue Culture and In VitroManipulation” in Corn and Corn Improvement 3d Edit. Sprague et al (eds)pp. 345-387 (American Soc. Of Agronomy 1988). The selectable markerincorporated in the DNA molecule allows for selection of transformants.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki et al, supra; Klein et al, Bio/Technology 10:268 (1992);and Weisinger et al., Ann. Rev. Genet. 22: 421-477 (1988). For example,the DNA construct may be introduced into the genomic DNA of the plantcell using techniques such as microprojectile-mediated delivery, Kleinet al., Nature 327: 70-73 (1987); electroporation, Fromm et al., Proc.Natl. Acad. Sci. 82: 5824 (1985); polyethylene glycol (PEG)precipitation, Paszkowski et al., Embo J. 3: 2717-2722 (1984); directgene transfer WO 85/01856 and EP No. 0 275 069; in vitro protoplasttransformation U.S. Pat. No. 4,684,611; and microinjection of plant cellprotoplasts or embryogenic callus. Crossway, Mol. Gen. Genetics202:179-185 (1985). Co-cultivation of plant tissue with Agrobacteriumtumefaciens is another option, where the DNA constructs are placed intoa binary vector system. Ishida et al., “High Efficiency Transformationof Maize (Zea mays L.) mediated by Agrobacterium tumefaciens” NatureBiotechnology 14:745-750 (1996). The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example Horsch et al., Science 233: 496-498 (1984),and Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1983).

Standard methods for transformation of canola are described by Moloneyet al. “High Efficiency Transformation of Brassica napus usingAgrobacterium Vectors” Plant Cell Reports 8:238-242 (1989). Corntransformation is described by Fromm et al, Bio/Technology 8:833 (1990)and Gordon-Kamm et al, supra. Agrobacterium is primarily used in dicots,but certain monocots such as maize can be transformed by Agrobacterium.U.S. Pat. No. 5,550,318. Rice transformation is described by Hiei etal., “Efficient Transformation of Rice (Oryza sativs L.) Mediated byAgrobacterium and Sequence Analysis of the Boundaries of the T-DNA” ThePlant Journal 6(2): 271-282 (1994, Christou et al, Trends inBiotechnology 10:239 (1992) and Lee et al, Proc. Nat'l Acad. Sci. USA88:6389 (1991)). Wheat can be transformed by techniques similar to thoseused for transforming corn or rice. Sorghum transformation is describedby Casas et al, supra and sorghum by Wan et al, Plant Physiciol. 104:37(1994). Soybean transformation is described in a number of publications,including U.S. Pat. No. 5,015,580.

It is preferred to select the highest level of expression of theprotease, and it is thus useful to ascertain expression levels intransformed plant cells, transgenic plants and tissue specificexpression. One such method is an ELISA assay which uses biotinylatedanti-trypsin or anti-trypsinogen polyclonal antibodies and astreptavidin-alkaline phosphatase conjugate. For example, an ELISA usedfor quantitative determination of trypsinogen or trypsin levels can bean antibody sandwich assay, which utilizes polyclonal rabbit antibodiesobtained commercially. The antibody is conjugated tostreptavidin-alkaline phosphatases for detection.

The levels of expression of the gene of interest can be enhanced by thestable maintenance of a protease encoding gene on a chromosome of thetransgenic plant. Use of linked genes, with herbicide resistance inphysical proximity to the trypsin or trypsinogen gene, would allow formaintaining selective pressure on the transgenic plant population andfor those plants where the genes of interest are not lost.

With transgenic plants according to the present invention, the proteasecan be produced in commercial quantities. Thus, the selection andpropagation techniques described above yield a plurality of transgenicplants which are harvested in a conventional manner. The plants can befed to the animal, or the protein extracted from plant tissue ofinterest or total biomass, depending on how the enzyme is to be used.Protease extraction from biomass can be accomplished by known methodswhich are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-96 (1981).

It is evident to one skilled in the art that there can be loss ofmaterial in any extraction method used. Thus, a minimum level ofexpression is required for the process to be economically feasible. Forthe relatively small number of transgenic plants that show higher levelsof expression, a genetic map can be generated, via conventional RFLP andPCR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, in Methods in Plant Molecular Biology andBiotechnology 269-84 (CRC Press 1993). Genetic mapping can be effected,first to identify DNA fragments which contain the integrated DNA andthen to locate the integration site more precisely. This furtheranalysis would consist primarily of DNA hybridizations, subcloning andsequencing. The information thus obtained would allow for the cloning ofa corresponding DNA fragment from a plant not engineered with aheterologous protease gene. (Here, “corresponding” refers to a DNAfragment that hybridizes under stringent conditions to the fragmentcontaining the protease encoding gene). The cloned fragment can be usedfor high level expression of another gene of interest. This isaccomplished by introducing the other gene into the plant chromosome, ata position and in an orientation corresponding to that of theheterologous gene. The insertion site for the gene of interest would notnecessarily have to be precisely the same as that of the trypsinogen ortrypsin gene, but simply in near proximity. Integration of an expressionvector constructed as described above, into the plant chromosome thenwould be accomplished via recombination between the cloned plant DNAfragment and the chromosome. Recombinants, where the gene of interestresides on the chromosome in a position corresponding to that of thehighly expressed protease gene likewise should express the gene at highlevels.

The following illustrates, but is not intended to limit the scope of theinvention. It will be evident to one skilled in the art that variationsand modifications are possible and fall within the scope and spirit ofthe invention.

Seed from the Westar variety of canola was transformed with constructscomprising elements according to the present invention, and have beendeposited with the American Type Culture Collection (ATCC) in Rockville,Md., under Accession no. 209942. Trypsinogen has also been introducedinto Hi-II maize plants. The constructs in question are designatedp4347, p5443 and p8244. The first construct comprises the ubiquitinpromoter, including the first exon and intron; the barley alpha amylaseexport signal sequence; a trypsinogen-encoding sequence; pinIIterminator; 35S promoter and terminator with the moPAT (maize optimizedPAT) selectable marker. The p5443 construct is the same, but with arepeating tandem trypsinogen encoding gene. The other constructcomprises a phaseolin promoter, brazil nut protein signal sequence, withthe trypsinogen encoding sequence and phaseolin terminator, all repeatedin tandem, along with the selectable marker, 35S promoter and terminatorand the PAT gene. The following provides further detail.

EXAMPLE 1 Isolation and Cloning of Trypsinogen Encoding DNA

The gene for cationic trypsinogen was cloned from bovine (Bos taurus)pancreas by the methods described here, with isolated RNA reversetranscribed into cDNA. The trypsinogen protein has 229 residues, withthe first 6 residues (VDDDDK-V=valine; D=aspartate; K=Iysine) beingcleaved to produce the active enzyme, trypsin. The Genbank accessionnumber is P00760. The sequence is set forth below. I CTTCATCTTTCTGGCTCTCT TGGGAGCCGC TGTTGCTTTC CCCGTGGACG 51 ATGATGACAA GATCGTGGGCGGCTACACCT GTGGGGCAAA TACTGTCCCC 101 TACCAAGTGT CCCTGAACTC TGGCTACCACTTCTGCGGGG GCTCCCTCAT 151 CAACAGCCAG TGGGTGGTGT CTGCGGCTCA CTGCTACAAGTCCGGAATCC 201 AAGTGCGTCT GGGAGAAGAC AACATTAATG TCGTTGAGGG CAATGAGCAA251 TTCATCAGCG CATCCAAGAG TATCGTCCAT CCCAGCTACA ACTCAAACAC 301CTTAAACAAC GACATCATGC TGATTAAACT GAAATCAGCT GCCAGTCTCA 351 ACAGCCGAGTAGCCTCTATC TCTCTGCCAA CATCCTGTGC CTCTGCTGGC 401 ACCCAGTGTC TCATCTCTGGCTGGGGCAAC ACCAAAAGCA GTGGCACCAG 451 CTACCCTGAT GTCCTGAAGT GTCTGAAGGCTCCCATCCTA TCAGACAGCT 501 CTTGCAAAAG TGCCTACCCA GGCCAGATCA CCAGCAACATGTTCTGTGCG 551 GGCTACCTGG AGGGCGGAAA GGACTCCTGC CAGGGTGACT CCGGTGGCCC601 TGTGGTCTGC AGTGGAAAGC TCCAGGGCAT TGTCTCCTGG GGCTCTGGCT 651GCGCTCAGAA AAACAAGCCT GGTGTCTACA CCAAGGTCTG CAACTACGTG 701 AGCTGGATTAAGGAGACCAT CGCCTCCAAC TAAATAGCTT CATCTCTTCA 751 TGACCCTCTC TGCTAGCCAGCTTCACCTTC CTCCCATCCT GAACGCACTA 801 CTTAAATAAA ATCATTTATA AAACC

EXAMPLE 2 Preparation of Plasmid p8244

The canola trypsinogen expression plasmid was generated by first addingbrazil nut protein signal sequence to the 5′ end of the trypsinogengene. An oligonucleotide was synthesized that contained the DNA codonsfor the brazil nut protein (bnp) transit peptide plus the codons for thefirst six amino acids of the trypsinogen protein. This oligonucleotidewas annealed to the trypsinogen cDNA and the second strand filled inusing PCR and the resulting fragment cloned into a pGEM vector. Thebnp/trypsinogen fusion was removed from the pGEM vector using therestriction enzymes Ncol and Hpal and cloned into another plasmid,between a phaseolin promoter and terminator. The entire transcriptionunit with promoter and terminator was cut from this intermediate vectorusing Eco RI and NotI and the overhanging ends filled in with the Klenowfragment of DNA Polymerase I, generating blunt ended inserts. The insertwas ligated into the filled EcoRI site of the binary vector pBIN19. Twoinserts of the trypsinogen gene ligated end to end in this vector,generating p8244, (FIG. 1) which contains two complete transcriptionunits for trypsinogen.

Preparation of Plasmid p4347

PCR mutagenesis of p8244 was conducted to add a HpaI site to the 3′ endof the coding region and the barley α-amylase signal sequence to the 5′end. The barley α-amylase sequence also contained a NcoI site at thestart of the coding region. The NcoI/HpaI fragment was cloned into anintermediate cloning vector which had been cleaved with BpuAI and HpaI.The resulting construct contained the ubiquitin promoter, thetrypsinogen open reading frame and the PinII terminator region. Theconstruct was then cleaved with NheI and NotI and cloned into the vectorp3770 which had been cleaved with NheI and NotI. This vector alsocontained the plant transcription unit comprised of the 35S promoter,the maize optimized PAT gene, and the 35S terminator region. The vectorwhich resulted, p4347, (FIG. 2) contained two plant transcription units:the ubiquitin promoter, trypsinogen gene, PinII terminator; and the 35Spromoter, the moPAT gene and the 35S terminator region.

Preparation of Plasmid p5443

PCR mutagenesis of p8244 was conducted to add a SalI site to the 5′ endand 3′ end of the coding region. The SalI site on the 5′ end wasdesigned to fuse the trypsinogen open reading frame in-frame with thetrypsinogen gene. The SalI fragment was cloned into a cloningintermediate vector which had been cleaved with SalI. The resultingconstruct contained an open reading frame containing two copies of thetrypsinogen gene. The NcoI/HpaI fragment from the construct was clonedinto a cloning intermediate vector which had been cleaved with BpuAI andHpaI. The resulting construct contained the ubiquitin promoter, thetrypsinogen open reading frame, and the PinII terminator region. Theconstruct which resulted, p5443 (FIG. 3) contained two planttranscription units: the ubiquitin promoter, double trypsinogen gene,PinII terminator; and the 35S promoter, the moPAT gene and the 35Sterminator region.

Preparation of Plasmid with Trypsin

Preparation of a plasmid will be carried out by the method describedabove, except that the trypsin gene instead of the trypsinogen gene willbe used.

EXAMPLE 3 Transformation of Maize

Fresh immature zygotic embryos were harvested from Hi-II maize kernelsat 1-2 mm in length. Fresh embryos were treated with 0.5 ml log phaseAgrobacterium strains carrying the superbinary vectors designed by JapanTobacco (Ishida, Y, H Saito, S Ohta, Y Hiei, T Komari and T Kumashiro.1996. “High efficiency transformation of maize (Zea mays L.) mediated byAgrobacterium tumefaciens”. Nature Biotechnology 14:745-750.). Bacteriawere grown overnight in a rich medium with appropriate antibiotics to anoptical density of 0.5, pelleted, then resuspended to the same densityin a microfuge tube in a standard liquid Murishige and Skoog mediumcontaining 100 μM acetosyringone. Embryos (5-1 0 per tube) weresonicated in the presence of the bacteria for 30 sec (Trick H and JFiner. 1997. “SAAT: sonication-assisted Agrobacterium-mediatedtransformation.” Transgenic Research 6-0.329-336), then plated on asolid medium as above. Embryos and bacteria were co-cultivated for 5days.

Embryos not subjected to sonication will be transferred to a bialaphosselective agent on embryonic callus medium and transferred thereafterevery two weeks to allow growth of transformed type II callus. Plantsare regenerated from the callus.

Transformation of Canola

Plasmid p8244 was introduced into Agrobacterium tumefaciens strainEHA105 by electroporation. This strain was co-cultivated with canolacotyledons as per the published method of Moloney et al. supra. Thismethod includes cutting petioles of 4.5 day germinated seedlings abovethe node, then dipping the base of the petiole in a suspension ofAgrobacterium diluted to 0.05 OD 600, co-cultivating without selectionfor 2-3 days at 25° C. and 100 μm² s¹ light intensity, then placing thecotyledons on selection medium containing 4 mg/L ppt(phosphinothricin—glufosinate ammonium). After 4-6 weeks on selection,shoots are removed and placed on rooting medium containing isobutyricacid. Rooted shoots are planted in soil and grown in the greenhouse forseed production.

EXAMPLE 4

Expression of trypsinogen in canola and corn was confirmed, andexpression level of this protein is 0.1%-1% of soluble protein fromcanola seed. The protein in canola has been assayed by Western blots, byELISA, and by activity assay as described below. Results are summarizedin Table 1. TABLE 1 Canola Transformation and Expression of TrypsinogenExpression Event Method Level I 1, T1 seed western 0.1% 1, T1 seedenzyme assay 0.15% 1, T1 seed quant. western 1.0% 1, T2 seed ELISA 0.18%1, T3 seed ELISA 0.4% 1, T3 seed western 0.1%

The corn tissue was analyzed by ELISA and transient expression oftrypsin at 0.19% total soluble protein confirmed. Stable expression willbe analyzed and is expected to confirm stable expression. Expressionlevels of 0.1% and higher are expected.

The Enzyme Linked Immunosorbent Assay was performed on canola usinganti-trypsinogen polyclonal antibodies and streptavidin alkalinephosphatase. The seed extracts were combined with buffering solution.After centrification and decanting, total protein concentration isassayed and adjusted to one concentration with PBST (phosphate buffersaline with 0.05% v/v polyoxyethylenesorbitan monolaureate (Tween-20)).Anti-trypsin antibodies were dispensed and incubated. After washing theplate with PBST, 10% normal rabbit serum and PBST were added andincubated. Trypsinogen standards were prepared and adjusted to the sameprotein concentration as the test samples. This was added to the wellalong with the test extracts. The plates were washed and diluted withbiotinylated anti-trypsinogen antibodies diluted with PBST and 10%normal rabbit serum and added. Following incubation, the plate waswashed and streptavidin-alkaline phosphatase conjugate diluted with PBSTand 10% normal rabbit serum added and incubated. The plate was washedand pNPP substrate solution added and incubated. The plate was read andamount of target protein calculated by interpolation from the standardcurve.

Activity of the enzyme was measured using a procedure in which antiserafrom four rabbits immunized with trypsinogen were pooled and the IgGfraction was purified using a Protein A agarose column. Proteinconcentration was determined using the A₂₈₀ spectrophotometric method.Fifty milligrams of pure IgG was buffer exchanged into coupling buffer(50 mM sodium acetate, pH 5.0) using 3 PD-10 columns, eluting in 10.5 mlcoupling buffer. 1.05 ml of 0.1M sodium periodate in water was added tothe IgG and the solution incubated for 1 hour at room temperature. Theunreacted periodate was removed by passing the solution over 4 PD-10columns equilibrated with coupling buffer. The oxidized antibody wasthen incubated overnight at 4° C. with 10 ml of hydrazide AX resin thathad been pre-equilibrated with coupling buffer. After coupling, thesupernantant is removed and checked for residual IgG. The resin was thenwashed sequentially with coupling buffer, washing buffer (PBS+0.5M NaCl)and PBST by resuspending in approximately 5 volumes of buffer followedby centrifugation to collect resin with a minimum of three repetitionsfor each buffer. The resin was then stored as a 1:1 suspension inPBST+0.1% thimersol. Samples containing trypsinogen were incubated with2511 of resin overnight to capture the enzyme.

These samples were activated with 1.0 U/sample enterokinase in 200 μlactivation buffer for one hour at room temperature with constant mixing.The activation mixture was removed and the samples washed with ≅1.0 mlof activation buffer and ≅1.0 ml of development buffer (0.1M TRIS, pH8.0) while under constant vacuum. Immediately after the removal of thedevelopment buffer wash, the samples were developed with 0.125 mMChromozyma® (TRY (Boehringer Mannheim Biochemicals) enzyme substrate indevelopment buffer at room temperature for 1 hour on a microtiter plateshaker. The resulting yellow supernatant is removed by gentle vacuum toa separate Dynatech microtiter plate and the samples were analyzed on aSpectroMax microtiter plate reader (Molecular Devices, Sunnyvale Calif.)at 405 nm. The concentration of trypsinogen in the sample is determinedby comparison to a standard curve.

Southern analysis is a well known technique to those skilled in the art.This common procedure involves isolating the plant DNA, cutting withrestriction endonucleases and fractionating the cut DNA on an agarosegel to separate the DNA by molecular weight and transferring tonitrocellulose membranes. It is then hybridized with the probe fragmentwhich was radioactively labeled with ³²P and washed in an SDS solution.Southern, E., or “Detection of a specific sequences among DNA fragmentsby gel electrophoresis” J. Mol. Biol. 98:503-517 (1975). Northernanalysis is also a commonly used technique by those skilled in the artand is similar to Southern analysis except that RNA is isolated andplaced on an agarose gel. The RNA is then hybridized with a labeledprobe. Potter, E. et al. “Thyrotropin releasing hormone exerts rapidnuclear effects to increase production of the primary prolactin mRNAtranscript” Proc. Nat. Acad. Sci. U.S.A. 78:6662-6666 (1981). A Westernanalysis is a variation of this technique, where instead of isolatingDNA, the protein of interest is isolated and placed on an acrylamidegel. The protein is then blotted onto a membrane and contacted with alabeling substance. (See e.g., Hood et al. “Commercial Production ofAvidin from Transgenic Maize; Characterization of Transformants,Production, Processing, Extraction and Purification” Molecular Breeding3:291-306 (1997))

Here, Western analyses were performed as described, with sampleselectrophoresed and the gel blotted followed by incubation overnightwith anti-trypsinogen IgG diluted in blocking buffer. The blot was thenwashed three times with PBST and incubated with 1:5000 dilution ofanti-rabbit peroxidase conjugate in blocking buffer and developed.

Western blots of trypsinogen positive canola seed are shown in FIGS. 4Aand B. In 4A the first six lanes show differing amounts of commercialtrypsinogen, used to provide a standard curve. The next three lanesrepresent extracts from transformed canola seeds: the first was extractfrom dry seed of a trypsinogen positive plant; the second was extractfrom seed of a trypsinogen positive plant that was imbibed in waterovernight; the third was extract from seed of a trypsinogen negativeplant, imbibed in water overnight.

FIG. 4B is another Western blot, with the first lane showing a controlof 15 ng of commercially produced trypsinogen, the second of controlseed extracted with PBST. The third lane is trypsinogen positive seedextracted with PBST, and the fourth is the seed extracted with a pH 5.6buffer. The fifth lane is control seed extracted with a pH5.6 buffer.The sixth lane is 150 ng of commercial mature trypsinogen, (i.e.,trypsin) followed by 50 ng of control trypsinogen. The eighth and ninthlanes are extract of transformed trypsinogen positive seed with theeighth imbibed in water and the last lane immunoprecipitated. In thethree bands of the canola seed extract, the terminal sequence of thecenter band matches mature trypsinogen.

A Southern analysis was performed as described, with DNA extracted froma To plant, digested with HindIII, and hybridized with the PAT gene as aprobe. The results are shown in FIG. 5A. The results show that one PCRpositive plant that showed resistance to glufosinate exposure hybridizedto the PAT gene. Five bands showed clear hybridization. FIG. 5B showsanother southern blot, of the T₃ progeny of the transformation eventused in the analysis shown in 5A. Fourteen of the progeny were screened.DNA was extracted, digested with PstI and hybridized with thetrypsinogen gene as a probe. After screening for glufosinate resistanceand by PCR, the Southern analysis showed four plants were positive fortrypsinogen. The restriction enzyme digests indicated three insertionsof the T-DNA occurred.

A Northern blot analysis will be performed on canola and is expected toreflect expression of trypsinogen.

EXAMPLE 5

The plasmid containing the trypsin gene will be introduced into corncells via the method described above, and expression determined usingELISA, western blot and activity assay. The analysis is expected toconfirm expression of trypsin in the plant. Levels of expression areexpected to be at 0.1% or higher.

Thus it is believed the invention accomplishes at least all of itsobjectives.

1. A transgenic plant comprising a DNA molecule comprising aheterologous nucleotide sequences coding for an active form of aprotease, wherein the protease is chymotrypsin operably linked to a seedspecific promoter to effect expression of the protease in the plant. 2.(canceled)
 3. A transgenic plant of claim 1 wherein the protease isexpressed at levels of at least about 0.1% of total soluble proteinexpressed in the plant.
 4. (canceled)
 5. The transgenic plant of claim 1or claim 3, wherein said heterologous nucleotide sequences furthercomprises a signal sequence that targets the protease to the cell wall.6. (canceled)
 7. The transgenic plant of claim 5 wherein the signalsequence is a barley alpha amylase peptide export signal sequence. 8.The transgenic plant of claim 1 wherein the plant is a corn plant. 9.Plant cells comprising a heterologous nucleotide sequences coding for anactive form of a protease, wherein the protease is chymotrypsin,operably linked to a seed specific promoter.
 10. (canceled)
 11. A methodof producing an active protease at levels of at least about 0.1% totalsoluble protein expressed in a plant, comprising providing biomass froma plurality of plants, of which at least certain plants contain a DNAmolecule comprising a heterologous nucleotide sequence coding for theprotease, wherein the protease is chymotrypsin, the nucleotide sequenceis operably linked to a promoter to effect expression of the protease,the promoter is seed specific, and extracting the protease from thebiomass, wherein the biomass comprises the seeds of the plants and theactive protease is produced at levels of at least about 0.1% totalsoluble protein expressed in the plant. 12-15. (canceled)
 16. The methodof claim 11 wherein the DNA molecule comprises a signal sequence thattargets the protease to the cell wall of the plants.
 17. (canceled) 18.(canceled)
 19. The plant cells of claim 9 wherein said heterologousnucleotide sequence further comprises a signal sequence that targets theprotease to the cell wall.